MCB_121_Lecture_05 - MCB 121 2010 Dr. Ted Powers Lecture 5:...

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Unformatted text preview: MCB 121 2010 Dr. Ted Powers Lecture 5: Protein Synthesis I Plan for Lectures 5-7: Lecture 5: tRNA and the genetic code Lecture 6: the ribosome and protein synthesis Lecture 7: translational regulation Reading for these lectures: Watson, Chapters 14 & 15 Problem Set 4: Requires reading the Problem Set 4 Supplement 1 MCB 121 2010 Dr. Ted Powers Lectures 5-7: Protein Synthesis ribosomes nascent peptide chain 5’ end of mRNA 3’ end of mRNA 2 Translation defines the genotype-phenotype transition genotype-----> phenotype DNA --> RNA --> Protein Protein synthesis Q: Why start a discussion of mechanisms of the central dogma with protein synthesis (versus replication, transcripton)? A: Translation links genotype and phenotype and is the most ancient of these processes 3 Goals for this lecture: I. Become familiar with the substrates of protein synthesis: mRNA and tRNA II. Enhanced appreciation for the genetic code 4 Messenger RNA (mRNA): generic features promoter Start ATG Stop TAG Trxn stop 5’ 5’ gene 5’ 5’UTR AUG ORF UAG 3’UTR mRNA 5’ UTR: 5’ Untranslated region 3’ UTR: 3’ Untranslated region ORF: Open Reading Frame = protein coding region 5 Messenger RNA: prokaryotes vs eukaryotes Prokaryotes: usually “polycistronic” (multiple ORFs) RBS AUG ORF 1 UAG RBS AUG ORF 2 UAG 5’ Problem: Multiple start AUGs. How to find them? Solution: Ribosomal Binding Site (RBS) aka “Shine-Dalgarno” site Base pairing between 16S rRNA of ribosome & mRNA: E. coli consensus: mRNA: 5’-GGAGG-3’ 16S rRNA: 3’-CCUCC-5’ 6 Messenger RNA: prokaryotes vs eukaryotes (cont.) Eukaryotes: 1. usually monocistronic (single ORF) 2. Two common post-transcriptional modifications 5’ m7G “Cap” & 3’ Poly A “Tail” Important for stability and regulation start AUG 5’UTR 5’m7G AUG ORF UAG 3’UTR AAAAAAAAA’-3 Problem: How to find the correct start AUG? Solution: ribosomal “scanning”; more about this later 7 Transfer RNA (tRNA): The “Adaptor” Two “Business ends” 1. Anticodon loop 2. Acceptor Stem Existence Predicted by Francis Crick in 1958, prior to their discovery. “Adaptor Hypothesis” Allows information flow Between two distinct chemical species:nucleotides & amino acids 8 Demonstrating the existence of a tRNA “adaptor” 1957: Paul Zamecnik and co-workers showed: (a) a small RNA can be coupled amino acids (b) the amino acid can be incorporated into protein Note: these researches didn’t know the tRNA was decoding mRNA, thus the true significance of their result was not appreciated! 9 tRNA Secondary Structure is defined by Complementary base pairing Allowed Base Pairs: G-C A-U G-U 10 However: There are several possible secondary structures How do we know which one is correct? Holley and co-workers 1965 11 Methods for RNA 2° Structure Predictions 1. Computer algorithms Based on global free energy ( G) minimizations e.g. maximize base pairing (esp G-C pairs) avoid loops with less than 3 bases Problem: usually wrong result Most recent iteration: “altRNA”: University of Washington of energy density and free energy of an RNA structure” Aksay et al., NAR 2007 “dynamic programming solution that minimizes the sum 12 tRNAPhe (Yeast) and the “altRNA” algorithm ACL CCA-3’ end 13 Methods for RNA 2° Structure Predictions 2. Phylogenetic Comparative Sequence Analysis Let nature do the experiment for you! How does it work? 14 Phylogenetic Comparative Sequence Analysis Is The Best Method for RNA 2°Structure Prediction Consider the same tRNA from three different species Organism X Organism Y Organism Z C-G C-G A-U G-C A-U C-G G-C A-U U-A A-U C-G C-G G-C G-C U-G Existence of compensatory base changes Provide evidence for the correct 2° structure 15 tRNA’s contain many modified nucleotides “D loop” “T C loop” 1. Important for 3-D folding 2. Contribute to translational accuracy 3. Cellular processes: tRNA nuclear export (eukaryotes) translation initiation 16 Origins of tRNA Tertiary Structure: The “L-shape” “D loop” “T C loop” 17 tRNA Tertiary Structure is stabilized by unusual base-base and base-backbone interactions 18 tRNA anticodon and the genetic code 19 Degeneracy of the genetic code •Triplet code consisting of 4 bases, so 43 = 64 possible codons •Only 20 amino acids and 3 stop codons- how can this be? •The code is degenerate: More than one codon for most amino acids Note: no codon codes for more than one amino acid •Mechanisms of degeneracy: 1. Isoacceptor tRNAs 2. “Wobble” Base Pairs 20 Degeneracy 1: Isoacceptor tRNAs definition: multiple tRNAs with different anticodons but code for the same amino acid. an Arg codon note: differences between codons usually at first and second position Example: Six Arginine codons; isoacceptors used 5’-CGU-3’ 1 2 3 tRNAArgI mRNA 3’-GCA-5’ ||| 5’-CGU-3’ tRNAArgIII mRNA 3-UCU-5’ ||| 5’-AGA-3’ 21 Degeneracy 2: Wobble Base Pairs definition: a single tRNA that can decode more than one codon note: this is specific for the third position of a codon Example: 2 Asp codons, only one tRNA an Asp codon 5’-GAC-3’ 1 2 3 tRNAAsp mRNA 3’-CUG-5’ ||| 5’-GAC-3’ tRNAAsp mRNA 3-CUG-5’ ||| 5’-GAU-3’ G-U Wobble Pair 22 There are Wobble “Rules” 5’ Base of tRNA Anticodon U C G A I 3’ Base of Codon Standard A G C U C Wobble G U U,A I = Inosine Formed by deamination of A Has properties of both A and G, accounting for its promiscuity 23 Inosine formed by deamination of Adenosine H2 O Adenine Inosine Guanine 24 Inosine at work Inosine rather than Adenosine is usually at the 5’ position of the anticodon tRNAArgII mRNA Arg codons 3’-GCI-5’ ||| 5’-CGC-3’ 5’-CGU-3’ 5’-CGA-3 25 Wobble Pairing Interactions 26 The aminoacyl acceptor stem-the other business end of tRNA Amino acyl linkage (Ester link) 3’-OH of ribose 27 During translation, the identity of the amino acid may not always be Inspected to ensure that it corresponds to the identity of the tRNA 1. Chemical reduction of Cys-tRNACys to Ala-tRNACys by Raney nickel 2. The tRNA is not otherwise altered and Ala is incorporated into protein Conclusion: tRNAs must be accurately “charge” with the correct aminoacid to avoid errors during protein synthesis. How is this accomplished? (Note that I will challenge this text book view in our next lecture) 28 tRNA aminoacyl synthetases (tRNA-RS) Connect tRNAs and their cognate amino acids The “Second Genetic Code”: Features: One tRNA-RS for each amino acid Thus, one RS will recognize different iso-acceptor tRNAs Energy of ATP hydrolysis used ensure that correct aminoacid and tRNA are matched. Components involved: tRNA, tRNA-RS, aa, ATP 29 tRNA aminoacyl synthetases(aka tRNA-RSs) Connect tRNAs and their cognate amino acids E. coli: 64 codons > 30 tRNAs 20 tRNA synthetases Two classes of tRNA-RS Structural and enzymatic differences Class I: GlnRS Class I: attach amino acid to 2’-OH Class II: attach amino acid to 3’-OH In both cases, energy of ATP hydrolysis used ensure that correct aminoacid and tRNA are matched. Process called the “second genetic code” Class II: AspRS 30 A Detailed View of a Synthetase-tRNA-amino acid-ATP quaternary complex Glutamyl-tRNA synthetase •Deep cleft for amino acid: allows screening based on size •Extensive contacts between tRNA and Synthetase. •These interactions include “Identity Elements” of tRNA used for recognition Especially acceptor stem and anticodon loop! 31 Two steps involved in tRNA aminoacylation 32 tRNA aminoacylation and editing: Pre-transfer versus post-transfer reactions RS + AMP + aa + tRNA Pre-transfer editing RS + ATP + tRNA + aa RS:tRNA:(AMP-aa) RS + aa-tRNA RS:aa-tRNA + AMP Post-transfer editing RS + aa + tRNA 33 The malleability of the genetic code Two general issues I. Suppression a) Missense b) Nonsense II. Evolution a) Naturally occuring alternate codes (e.g. human mitochondria) b) Biotechnology and codon-expansion Underlying theme Dynamic relationship between codon-anticodon interactions 34 Amber mutants = UAG stop codon Cause premature Termination of translation Other stop codons: UAA = Ochre UGA = Opal 35 The fragility of the genetic code: Suppressor tRNAs Q: How do you translate a full length protein that contains an amber amber (UAG) nonsense mutation? A: Nonsense suppressor tRNA Step 1 5’-UGG-3’ Trp (WT) 5’-UAG-3’ Amber stop Step 2 3’-AUG-5’ 3’-AUC-5’ tRNATyr Amber suppressor Step 3 3’-AUC-5’ tRNASup ||| 5’-UAG-3’ Amber stop 36 Experiment that demonstrated nonsense suppression WT gene has two activities:”A” and “B” 37 Important Caveats for non-sense suppression to work in a cell I. Redundant (multiple copies) of tRNAs needed One tRNA must remain WT to decode normal “Sense” codon The other tRNA can be mutated to decode the “Nonsense” codon 3’-AUG-5’ tRNATyr 3’-AUC-5’ Amber ||| suppressor 5’-UAG-3’ 3’-AUG-5’ ||| 5’-UAC-3’ tRNATyr Tyr codon 38 Important Caveats for non-sense suppression to work in a cell II. Inserted amino acid must yield a functional protein III. Non-sense suppression must be relatively inefficient. Why? Normal stop codons must still be recognized by Release Factors Thus, if non-sense suppressors were too efficient, normal stop codons would be replaced by aminoacids This could be toxic for cells Amber suppressors (UAG): 10-50% efficient but UAG is rarely used as a stop codon Ochre suppressors (UAA): <10% efficient and used frequently Opal suppressors (UGA): very inefficient (<1%) Why is the Opal suppressor so inefficient? 39 Opal suppressor tRNA has a WT anticodon! Opal suppressor tRNATrp Mutation G --> A at position 24 In D stem 3’-ACC-5’ tRNATrp Suppressor ||| 5’-UGA-3’ Opal nonsense Position of mutation Thus, A-C wobble is occuring in this case! 40 Next time: How does protein synthesis work? Pictured: Actively translating Polyribosomes 41 ...
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This note was uploaded on 09/23/2010 for the course NPB 8746546 taught by Professor Goldberg during the Spring '10 term at UC Davis.

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